Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting a wide variety of mammals including sheep and goats (scrapie), cervid spp. (chronic wasting disease), and humans (Creutzfeldt-Jakob disease). Our studies are focused on the prion protein (PrP) due to the critical role of this protein in controlling many aspects of TSE pathogenesis such as susceptibility to disease and interspecies transmission. A central event in TSE disease involves the conversion of the normal host cellular prion protein (PrPC) to a partially protease-resistant, aggregated, disease-associated isoform (PrPSc). TSE-induced pathology is usually associated with PrP-res deposition, but the mechanism of neurodegeneration is not understood. The nature of the infectious agent, called a prion, remains uncertain but is thought to be composed primarily of misfolded PrP, perhaps in complex with another host accessory molecule(s). PrPC is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein, and the majority of PrPSc produced in vivo contains this GPI anchor. Membrane association of both normal and disease-associated PrP isoforms may influence many features of prion disease and PrPC function. Our work is focused on elucidating mechanisms of uptake, replication, and spread of prions, in addition to determining the biochemical composition of mammalian prions. In 2012, we have: 1) shown that purified recombinant PrP can be converted into a TSE agent, providing insight into the biochemical composition of prions; 2) created novel strains of TSE agents using recombinant PrP by inoculation into mice expressing GPI-anchorless, showing that GPI anchoring of host PrPC can modulate the selection of these synthetic prion strains; 3) further investigated mechanisms of intercellular spread of PrPSc via imaging studies in novel neuronal culture systems; and 4) identified new cell types that avidly internalize PrPSc particles and thus may play a role in early events during TSE infection of animals and humans.